1. Field of Invention
The present invention generally relates to blood chemistry laboratory techniques and apparatus. More particularly, the present invention relates to methods for separating mononuclear cells, such as lymphocytes and monocytes, from whole blood specimens, especially assemblies or methods which maintain blood in an anticoagulated state prior to partitioning the sample into discrete layers using centrifugation. Additionally, the present invention relates to blood separation assemblies which provide a high recovery of mononuclear cells without significant contamination by red blood cells.
2. Description of the Prior Art
Considerable research has been conducted in recent years to develop improved methods and devices for separating mononuclear cells, such as lymphocytes and monocytes, from whole blood. Effective separation and isolation of these cells is often critical to various clinical assays as well as to research laboratory protocols. Consequently, a variety of blood collection/separation tubes have been developed.
For example, one currently available blood separation device includes a blood collection tube containing an aliquot of a Newtonian gel and an aliquot of a liquid density medium, such as Ficoll-Paque.TM.. The Newtonian gel acts as a barrier between the liquid density medium and a sample of anticoagulated whole blood which is placed in the tube above the gel prior to centrifugation. When the tube is subsequently centrifuged, the liquid density medium acts to separate the mononuclear cells from the other blood components. The separation occurs because the specific gravity of the density medium is greater than the specific gravity of the mononuclear cells but less than the specific gravity of the other blood components.
The aforementioned device and method works well where the tube is immediately centrifuged upon receipt of the whole blood sample but cannot be utilized as an effective separation means where the blood sample is placed in the tube and must be shipped elsewhere for subsequent centrifugation, isolation and analysis. The reason for this limitation is that both the Newtonian gel and the liquid density medium are flowable liquids and will not retain their respective positions in the tube due to movements occurring during shipping. As a result, the blood sample will mix with the density medium during shipping, thereby diluting it. Dilution of the medium adversely affects the proper separation of the blood sample upon subsequent centrifugation at the laboratory. Such dilution is especially problematic in laboratory studies where contamination of the mononuclear layer by red blood cells must be minimized.
Consequently, other blood separation devices have been developed. For example, one of these devices is a blood separation tube in which a porous foam-like plug is deployed above a liquid density separation medium. The plug functions as a "baffle" for preventing a blood sample introduced into the tube from mixing with the separation medium prior to centrifugation. Although the plug is constructed with a porosity suitable for allowing migration of red blood cells and separation medium therethrough during centrifugation, the plug design prevents the blood sample from interfacing with the separation medium prior to centrifugation, thereby facilitating a cleaner separation of the mononuclear cell layer.
Another currently available blood separation device utilizes a non-Newtonian, thixotropic gel. This gel is positioned within a blood collection tube in order to form a stable barrier between the liquid density medium and the blood sample prior to centrifugation. For example, U.S. Pat. No. 4,867,887 to Smith discloses several embodiments of a separation device utilizing a thixotropic gel. In one such embodiment, a blood collection tube includes a layer of a Newtonian gel positioned in the bottom of the collection tube. A thixotropic gel is temporarily positioned immediately above the Newtonian gel in order to prevent the Newtonian gel from mixing with the blood sample prior to centrifugation. A space is provided above the thixotropic gel suitable for allowing placement of a blood sample into the tube prior to centrifugation.
Upon the addition of an anticoagulated whole blood sample and subsequent centrifugation, the thixotropic gel moves to the bottom of the tube, thereby displacing the Newtonian gel. Since the specific gravity of the Newtonian gel is less than the specific gravity of the thixotropic gel, the Newtonian gel moves to a new position above the thixotropic gel where it mixes with the blood sample and acts as a density separation medium much like the liquid density medium, Ficoll-Paque.TM.. Red blood cells, which have the highest specific gravity of any of the components in whole blood, are pelleted along with the granulocytes to the bottom of the tube immediately below the thixotropic layer. The mononuclear cells form a layer immediately above the Newtonian gel, thereby rendering an effectively isolated layer of cells to be removed for clinical analysis or other laboratory manipulations.
A further embodiment disclosed in U.S. Pat. No. 4,867,887 to Smith involves a similar configuration for a blood separation device including a collection tube containing a density gradient material, a Newtonian gel, a thixotropic gel and an aliquot of an anticoagulant/cell-sustaining solution. Prior to the addition of any blood sample or centrifugation, the density gradient material and the Newtonian gel are positioned in the bottom portion of the tube immediately below the thixotropic gel. An aliquot of anticoagulant/cell-sustaining solution is placed into the tube above the thixotropic gel which serves as a temporary barrier to isolate the solution from the other components in the tube. A space is provided within the tube above the solution. The space is suitable for allowing the placement of an anticoagulated blood sample into the tube prior to centrifugation.
After a sample has been received in the tube and centrifugation is performed, the red blood cells and granulocytes are pelleted to a position at the bottom of the tube immediately below the thixotropic gel layer. The density gradient material moves to a new position above the thixotropic gel where it mixes with the blood sample as well as the anticoagulant/cell-sustaining solution in order to perform a separation function. The density gradient material includes both a light and a heavy phase which separate into two layers in order to sandwich the Newtonian gel layer therebetween. The mononuclear cells form a layer immediately above the lighter phase of the density gradient and are subsequently removed for analysis.
In both of the embodiments disclosed by Smith, the thixotropic gel acts as a barrier to isolate the other components of the separation device from one another prior to centrifugation. In the second embodiment, the Newtonian gel is not used as a barrier nor a separation medium since the thixotropic gel and the density gradient material perform these functions, respectively. The Newtonian gel alternatively performs a quality control function in that it provides a sticky surface to retain any residual red blood cells located near the mononuclear cell layer after centrifugation, thereby facilitating removal of the mononuclear cells and limiting any contamination with residual red blood cells.
U.S. Pat. No. 3,852,194 discloses a simpler blood separation device utilizing a thixotropic gel material having a specific gravity in between that of mononuclear cells and other components of whole blood, such as red blood cells and granulocytes. Consequently, the thixotropic gel functions as a separation medium rather than performing a barrier function as in the '887 patent to Smith.
Other blood separation devices are designed for blood collection as well as separation. For example, various direct draw blood collection containers are currently available. Some of these are designed for undergoing subsequent centrifugation, thereby eliminating the need for mechanically transferring a blood sample to a different containier prior to inducing separation via centrifugation.
The blood collection and/or separation tubes mentioned above are only a few of many such containers known to those skilled in the art. Regardless of the type of container employed to perform the collection and/or separation function, preventing contamination of the mononuclear cell layer with red blood cells remains a key concern for diagnostic studies which demand minimal levels of RBC contamination.
Various anticoagulants have been used in blood collection/separation devices either alone or in conjunction with a cell-sustaining solutions in order to preserve the blood sample in an uncoagulated state for a period of time prior to centrifugation and analysis. For example, some common anticoagulants include sodium heparin, K.sub.3 EDTA and sodium citrate. In particular, sodium citrate solutions have been used for many years as anticoagulants. For example, current requirements for gene amplification technologies, such as the polymerase chain reaction, recommend the use of sodium citrate for performing an anticoagulation function in whole blood. See Holodniy, M.; Kim, S.; Katzenstein, D.; Konrad, M.; Groves, E.; Merigan, T. C.; "Inhibition of Human Immunodeficiency Virus Gene Amplification by Heparin", J. Clin. Microbiol. 29:676-679 (1991).
It is known that calcium plays a key role in the blood coagulation cascade. Sodium citrate solutions prevent the participation of calcium in blood coagulation. Typically, these sodium citrate solutions are added to freshly collected whole blood to prevent coagulation. Subsequently, calcium can be added back to the whole blood suspension to induce subsequent coagulation when desired.
Sodium citrate is a particularly advantageous anticoagulant as it provides good buffering capabilities over a range of pH. In particular, the buffering capability of sodium citrate is attributable to three carboxyl groups present on the corresponding acid of the compound. Since sodium citrate is the corresponding sodium-based salt of citric acid, it is actually the citric acid/sodium citrate combination that actually functions to perform the buffering chemistry.
As mentioned above, citric acid (hydroxytricarboxylic acid) has 3 carboxyl groups and consequently 3 pKa's. The first pKa appears at a pH of about 3.06. The second pKa appears at a pH of about 4.74. The third pKa appears at a pH of about 5.4. Accordingly, sodium citrate performs its most effective buffering functions at these pH values and is especially useful in performing buffering functions when added to in vitro cell suspensions. Consequently, sodium citrate has been used as an anticoagulant in a variety of blood separation devices due to its buffering capability over a range in pH.
In particular, citrate has been commonly used as an anticoagulant in three types of solutions. The first type of solution is referred to as buffered sodium citrate. The second type of solution is typically referred to as CPD solution or citrate-phosphate-dextrose. The third type is denoted as ACD or acid-citrate-dextrose. The citrate ion concentration in these solutions is typically greater than the concentration needed to perform an anticoagulation function. Examples of these three solutions as well as other currently available citrate-based anticoagulant solutions are included in the following table.